J. AMER.SOC.HORT.SCI. 134(4):479–487. 2009. Structure and Histochemistry of the Micropylar and Chalazal Regions of the Perisperm–endosperm Envelope of Cucumber Seeds Associated with Solute Permeability and Germination Yuliya A. Salanenka, Martin C. Goffinet, and Alan G. Taylor1 Department of Horticultural Sciences, NYSAES, Cornell University, 630 W. North Street, Geneva, NY 14456 ADDITIONAL INDEX WORDS. callose, Cucurbitaceae, weakening, nucellar beak, pectin, cuticle, suspensor remnant ABSTRACT. The perisperm–endosperm (PE) envelope surrounding the embryo of cucumber (Cucumis sativus) acts as a barrier to apoplastic permeability and radicle emergence. The envelope consists of a single cell layer of endosperm whose outer surface is covered by noncellular lipid and callose-rich layers. We compared the structure and histochemistry of the radicle tip and chalazal regions of the envelope, because these regions differ in permeability. Seeds were treated with coumarin 151, a nonionic, fluorescent tracer with systemic activity. Treated seeds were imbibed and on seedcoat removal, the root tip area of the membrane-covered embryo accumulated the fluorescent tracer, but the tracer could not penetrate the envelope that bordered the cotyledons and chalazal region. The cone- shaped remnant of tissue opposite the micropylar region of the envelope was identified as nucellar tissue, the ‘‘nucellar beak.’’ The cuticular membrane and callose layer of the PE envelope were interrupted in the nucellar beak as well as in the chalazal region. Their role in permeability is apparently substituted by the presence of thick-walled suberized cells in the beak and chalaza. A canal was observed in the center of the nucellar beak that likely provided a conduit for the tracer to diffuse from the environment to the embryo. This canal was the remnant of pollen tube entry through the nucellus and was plugged with several cells, presumably residue of the suspensor. These cells degenerated just before cucumber seed germination. This remnant of the pollen tube canal presumably offers less mechanical resistance in the nucellar beak that might help facilitate radicle protrusion during germination. Cells of the outermost and basal regions of the nucellar beak as well as the walls of endosperm cells contained pectic material. Significant pectin methylesterase activity was found in the lateral and cap regions of the PE envelope long before seed germination. Lack of callose in the envelope at the radicle tip suggests that callose does not act as a barrier to radicle emergence during cucumber seed germination. Cucumber is a species whose seeds have a semipermeable (Ramakrishna and Amritphale, 2005; Welbaum and Bradford, barrier restricting transport of solutes. A thin membrane beneath 1990; Yim and Bradford, 1998), attention was not paid to the testa, the perisperm–endosperm (PE) envelope, acts as particular regions of the PE envelope. This article focuses on a barrier to apoplastic permeability of cucumber and other the micropylar and chalazal regions of the PE envelope in Cucurbitaceae seeds (Ramakrishna and Amritphale, 2005; Yim cucumber seed. These two regions are positioned closely to one and Bradford, 1998). This envelope is reported to consist of another in the fully anatropous ovule (Tillman, 1906), and these a single cell layer of endosperm whose outer surface is covered regions provide specialized functions in Cucurbitaceae seeds. by lipid and callose layers (Yim and Bradford, 1998). The PE The chalazal region of the PE envelope, located opposite the envelope of seeds of the genus Cucumis limits transport of micropyle, is involved in transport of nutrients from maternal tetrazolium salts, abscisic acid (Ramakrishna and Amritphale, tissue into the developing embryo during seed development. 2005), and prevents solute leakage from dead seeds (Welbaum The micropylar region acts as a barrier for radicle emergence and Bradford, 1990; Yim and Bradford, 1998). We have es- during germination and is studied in many species such as tablished that the cucumber seed envelope is impermeable to tomato (Solanum lycopersicum) (Groot and Karssen, 1987; the nonionic, moderately lipophilic, fluorescent tracer, 7-amino- Nonogaki and Morohashi, 1996), lettuce (Lactuca sativa)(Ikuma 4-(trifluoromethyl) coumarin (coumarin 151) (Salanenka and and Thimann, 1963), pepper (Capsicum annuum) (Watkins et al., Taylor, 2008), except in the region that covers the radicle tip 1985), and tobacco (Nicotiana tabacum) (Leubner-Metzger area (Fig. 1A). Tao and Khan (1974) observed the permeation et al., 1995, 1996). In seeds of Cucurbitaceae, mannan-rich of chemicals into this radicle tip area in Cucurbita maxima endosperm and a callose layer of the PE envelope are con- seeds, although they did not study its causal factor. Tao and sidered to form the primary barrier to radicle emergence during Khan (1974) suggested that the anatomical structure of the tip germination. Weakening of the mannan-rich endosperm region of the seed might relate to enhanced permeability. and callose in the micropylar region is necessary for radi- Although the structure of the PE envelope was studied cle protrusion through the PE envelope (Ramakrishna and Amritphale, 2005; Welbaum et al., 1995, 1998). In the present work, we focus on the structure and histo- Received for publication 14 Jan. 2009. Accepted for publication 19 Aug. 2009. Partial funding was provided by the American Seed Research Foundation and chemistry of the micropylar and chalazal regions of the PE Multi-State project W-1168. envelope of cucumber seed in relation to solute permeability 1Corresponding author. E-mail: [email protected]. and germination. J. AMER.SOC.HORT.SCI. 134(4):479–487. 2009. 479 Materials and Methods sections. Sections in staining solution were mounted on glass slides, covered with coverslips, and then observed in visible Plant material radiation. Red–violet color indicated lignin (Jensen, 1962). Mature seeds of ‘Vlaspik’ cucumber were obtained from LIPIDS. Saturated solutions of either Sudan black B or Seminis, Inc. (Oxnard, CA) and were stored in plastic con- a Sudan III–IV mixture in 70% (v/v) ethanol were applied to tainers at 4 °C until needed. For embryological studies, seeds tissue sections and incubated up to 30 min. Cutin and suberin were collected from cucumber fruit at different stages of fruit give a red–orange color with Sudan III–IV and a blue–black development. Seeds were removed from fruit and washed color with Sudan black B (Krishnamurthy, 1999). For fluores- vigorously in water to remove residual mucilaginous fruit cent microscopy of lipids, neutral red (Lulai and Morgan, tissue. Excess water was removed, and seeds were fixed for 1992), rhodamine B, and auramine O (Gahan, 1984) were microscopy as described subsequently. applied to sections. Sections were stained with a 0.1% solution of neutral red in 0.1 M potassium phosphate, pH 6.5, for 1 min, Permeability test and gross anatomical observations rinsed briefly in buffer, and observed under blue–violet Agarose was added to deionized water saturated with excitation. Other sections were covered with a 0.1% (w/v) fluorescent tracer (coumarin 151) and microwaved. The solu- rhodamine B aqueous solution for 30 min and then mounted tion (0.6% w/v) was poured into 9-cm petri dishes and cooled at with a coverslip using distilled water. Specimens were observed room temperature. Seed coats were carefully removed, em- under ultraviolet excitation. Auramine O (0.01% solution) pre- bryos immersed in the solidified agarose gel, and incubated at pared in 0.05 M Tris-HCl buffer, pH 7.2, was applied to sections 25 °C for 12 h to study perfusion of tracer through the PE for 10 min, coverslipped, and viewed with blue–violet excitation. envelope. Seeds imbibed in the agarose gel were sectioned and TANNIN. A drop of saturated alcoholic vanillin was placed examined under low magnification (·3 and ·10) with a long- on fresh hand-cut sections, and several drops of concentrated wavelength ultraviolet radiation source (365 nm). For gross HCl were added. Immediate development of a bright red color seed anatomy, seeds were decoated and observed without indicated the presence of tannins (Gardner, 1975). further preparations with a stereomicroscope (SZX12; Olym- NONESTERIFIED PECTIN. Ruthenium red, Alcian blue 8GX, pus, Center Valley, PA) equipped with a SPOT Insight camera and toluidine blue O were used to identify nonesterified pectin and software (Version 4.5; SPOT Imaging Solutions, Sterling and other acidic polysaccharides. Sections were flooded with Heights, MI). a 0.05% aqueous solution of ruthenium red for 20 min and observed for red color (Jensen, 1962). A 1% Alcian blue 8GX Tissue preparation and fixation for microscopy solution in 3% acetic acid (pH 2.5) was applied to tissue Mature dry seeds were fixed in a chromic acid–acetic acid– sections for 20 min, rinsed with water, and the sections were formalin mixture (CRAF III) and subsequently dehydrated and examined for blue color under visible radiation (Clark, 1981; cleared in an ethanol–tertiary butanol (TBA) series as described Lev and Spicer, 1964). For metachromatic staining, a 0.05% by Ruzin (1999). Briefly, seeds selected for soundness and toluidine blue O solution, prepared in citrate-phosphate buffer uniformity were fixed in CRAF III solution at room temperature (pH 4.4), was applied to sections for 30 s before paraffin for 12 h. The fixative was then rinsed in running water removal from sections (Sakai, 1973). Slides were rinsed in overnight. After dehydration in a graded ethanol-TBA series, water for 2 min and air-dried. Paraffin was then removed with followed by infiltration by absolute TBA, the specimens were xylene and sections were examined for red–purple coloration, embedded in a paraffin mixture (Tissue Prep; Fisher Scientific, indicating acidic polysaccharides. Polyphenols were stained Fair Lawn, NJ). Tissue sections (7 mm) then were cut with green–blue or dark blue. a steel knife on a rotary microtome. Thereafter, tissue sections CELLULOSE. Sections were stained with a 0.1% (w/v) were processed for microscopic observations based on specific aqueous solution of calcofluor white M2R for 1 min and needs.